KR102106589B1 - Microfluidic aliquoting for single-cell isolation - Google Patents

Abstract

A microfluidic aliquoting (MA) chip designed to be mounted on a petri dish according to the present invention generally includes a central well (entrance) connected to a plurality of lateral wells (outlets) by a plurality of microchannels. A relatively large dose (eg, 120 μL) of cell suspension containing several cells can be injected into the inlet of the MA chip, and single cells have a time of less than 1 minute by positive pressure-driving flow. Can be distributed substantially simultaneously and uniformly into multiple (eg, 120) lateral wells containing single cells within. The MA chip has high cell regeneration efficiency. The MA chip has advantages such as rapid separation and easy identification of single cells, high cell viability, high enrichment factor, and convenient migration of submicroliter single cell suspensions, thus separating CTC from blood, single It can be easily applied to cell cloning, PCR, and sequencing.

Description

MICROFLUIDIC ALIQUOTING FOR SINGLE-CELL ISOLATION

Some embodiments of the invention broadly relate to methods and devices for single cell isolation (e.g., for treatment or analysis), more specifically to isolate single cells using microfluidic technology It's about technology.

Microfluidics is a comprehensive discipline that encompasses engineering, physics, chemistry, biochemistry, nanotechnology, and biotechnology, which processes small volumes of fluids to realize multiplexing, automation, and high-throughput screening. It is practically applicable to system design. The development of microfluidic technologies includes molecular biological processes for enzymatic analysis (e.g. glucose and lactic acid analysis), DNA analysis (e.g. polymerase chain reaction (PCR) and rapid sequencing) and protein genetics (proteomics). Innovatively changing. The basic idea of microfluidic biochips is to integrate analytical tasks such as detection as well as sample preparation and sample preparation on one chip.

There is increasing evidence that the heterogeneity of phenotypes and genotypes is widespread in cell populations. The main information of rare individual cells may not be revealed by mass cell analysis. Thus, single cell analysis, especially DNA and RNA sequencing, is of great importance for clonal mutation, tumor evolution, embryonic development and immunological studies.

The initial and key steps for downstream single-cell genetic analysis are effectively separating the desired living single cells from heterogeneous cell populations in a submicroliter volume medium and performing PCR analysis on them (PCR Is a molecular biological technique used to amplify one or more copies of a DNA fragment by a few digits to produce thousands to millions of copies of a particular DNA sequence).

In addition to laser capture microdissection, which is mainly used to separate single cells from formalin-fixed paraffin-embedded tissue, four main approaches for isolating single cells from current cell suspensions Is used.

-The most commonly used method, serial dilution, has been widely applied for colony formation, but is not suitable for PCR analysis where a single cell must be separated into a submicroliter suspension for better amplification reactions. . Nevertheless, these colonies are still needed for further analysis to determine whether these colonies originate from a single cell, which is very difficult to accurately calculate after inoculation in a 96 well culture dish.

-Micromanipulation was developed primarily to isolate single cells for genome / transcriptome sequencing. However, due to the long process time and low throughput, it is difficult to meet the increased requirement of quickly preparing tens or even hundreds of single cells 10. In addition, sucking up a single cell using the researcher's mouth can rely heavily on personal skills.

-Flow cytometry is a well-established method that is particularly well suited to classifying specific cells with high efficiency based on a preset fluorescence gating strategy, but it is difficult to maintain high single cell viability, and is rare. It cannot be applied to a limited number of cells, such as clinical samples.

-Because microchannel and cell size are similar, recently developed microfluidic technology provides an important method for single cell manipulation. However, the potential for microfluidic technologies, such as requiring additional techniques for microfluidic manipulation, lack of compatibility with existing experimental platforms, and the inability to selectively recover single cells isolated from the microchip for further analysis is difficult or difficult. Disadvantages greatly limit its application in general laboratories.

U.S. Patent No. 6632656 (2003-10-14; Thomas et al.), Incorporated herein by reference, describes apparatus and methods for performing cell growth and cell-based assays in liquid media. The apparatus includes a base plate supporting a plurality of micro-channel members (each of the micro-channel members includes a cell growth chamber, an inlet channel for supplying a liquid sample, and an outlet channel for removing liquid sample from the inlet channel) box); And a cover plate located over the base plate to form the chambers and connection channels (the cover plate has holes for providing access to the channel). Means for cell attachment and cell growth are included in the cell growth chamber. Hereinafter, it will be described in more detail below with reference to the drawings:

As shown in FIG. 1B, the device is rotated to provide a sample inlet connected to an annular sample reservoir 9 positioned toward the center of the disc and connected to a plurality of radially dispersed microchannel analysis members 6 in turn. A disk 18 is possible, and each of the microchannel members comprises a cell growth chamber, a sample inlet channel, an outlet channel for removing liquid from the sample inlet channel, and the disk for forming closed chambers and connecting channels. It includes a cover plate located thereon. One end of each of the microchannel members is connected to the central sample reservoir 9, and an end opposite the end is connected to a common waste channel 10.

Each of the radially-dispersed microchannel members 6 (shown in FIG. 1A) of the microfabricated device is a sample inlet channel 1 connected to a reservoir 9 located at its left end, cell growth and channel ( It includes a cell growth chamber 2 connected to the analytical chamber 3 through 4), and an outlet channel 5 connected to the right end of the waste channel 10.

Suitably, the disk 18 consists of a one or two piece mold structure, and any transparent plastic or polymer by separate molds assembled together to provide a closed structure with openings at predetermined locations. Formed from a material, it is possible to inject a liquid into the device and remove the waste liquid from it. In its simplest form, the device is manufactured as two complementary parts, and one or each of the two parts includes molding structures that, when secured together, form a series of interconnected microchannel members within the body of the solid disk. .

Alternatively, the microchannel members may be formed by a micro-machining method in which the microchannels and chambers forming the microchannel members are finely processed within the surface of a disc, and a cover plate (eg, a plastic film) ) Is attached to the surface to surround the channels and chambers.

The size of the device will depend to some extent on the application. That is, the device will be of a size suitable for use with eukaryotic cells. This imposes a lower limit on all channels designed to allow cell migration and determines the size of the cell inhibition or growth region depending on the number of cells present in each assay. On average, mammalian cells growing in the medium have an area of ˜300 μm ² , and non-adherent cells and non-adherent adherent cells have a spherical diameter of ˜10 μm. Consequently, channels for the movement of cells in the device are likely to have a size of approximately 20 to 30 μm or larger. The size of the area occupied by the cells depends on the number of cells required to perform the analysis (the number of cells is determined by sensitivity and statistical needs). It is believed that a typical analysis requires a minimum of 500 to 1000 cells, and for this, a structure of 150,000 to 300,000 μm ² for adherent cells, ie, a circular “well” having a diameter of about 400 to 600 μm.

The microchannels are preferably simultaneously injected into the cell growth chamber by injecting the sample reservoir into a cell suspension in a liquid medium through a sample inlet, after which the cell suspension is moved outward toward the outer periphery of the disk by centrifugal force. The disk 18 is configured to be rotated by suitable means at a speed sufficient to be described below. The cell suspension is distributed along each of the inlet microchannels 1, 8 towards the cell growth chambers 2, 7 due to the movement of the liquid. The rotational speed of the disc provides sufficient centrifugal force to allow the liquid to flow to fill the cell growth chamber (2, 7), but the liquid has a small diameter located on the opposite side of the cell growth chamber (2, 7). It does not provide enough force to enter the restricted channels 4, 16.

It is an object of the present invention to provide a simple, fast and versatile single cell separation technique (method and apparatus).

Generally, aliquoting means taking a representative sample from a large amount of material (usually a liquid). In this case, an aliquot means separating a single cell from a suspension with multiple cells. This process is called "microfluidic aliquoting" because it is done in a liquid environment using microfluidic technology.

According to the present invention, microfluid fraction (MA) chips (or substrates, "MA chips", "MA-chips") or modified versions thereof, which can generally have a disc shape and can be fabricated to fit into a Petri dish It may be referred to as) may include a central well (entrance) connected to a plurality of side wells (or outer wells, outlet) by a plurality of micro-channels (channels). In operation, a cell suspension containing a small number of cells (e.g., about 2 to 120 cells) can be injected into the inlet of the MA chip (using a pipette to push the central well by static pressure), and single cells They can be moved to a portion of the side wells (outer wells) by a constant pressure drive flow through the microchannels.

According to some embodiments (examples) of the present invention, a microfluidic fractionation (MA) chip for single cell separation comprises a disk-shaped chip having a radius, center, top surface, bottom surface, outer edge, and thickness; A single central well (entrance) disposed substantially at the center of the chip, extending to the top surface of the chip, and accessible from the top surface of the chip; A plurality of side wells (outlets) disposed in an annular portion of the outer shell of the chip, extending to the upper surface of the chip, and accessible from the upper surface of the chip; And a plurality of channels extending from the bottom surface of the chip and extending from the center well to the side wells in fluid communication with the center well and the side wells.

The chip is selected from the group consisting of polydimethylsiloxane (PDMS), poly (methyl methacrylate), PMMA, polystyrene (PS), and polycarbonate (PC). And a thin sheet made of a flexible or semi-rigid material. The chip may have a thickness of about 1 mm, may have a size and shape that can be mounted in a Petri dish, and may have a radius of about 3 cm. The center well may extend to pass completely through the chip; The side wells may extend to pass completely through the chip.

The center well may have a diameter of about 3 mm and a volume of about 4 μl. At least some of the side wells may have a diameter of about 1.5 mm and a volume of about 1 μl.

The first set of the plurality of side wells may be located at a first radius r1 from the center of the chip, and the second set of the plurality of side wells may be located at a second radius r2 from the center of the chip. can do. The second radius r2 may be larger than the first radius r1. The first set and the second set of side wells may be interleaved such that the second set of side wells is located at a circumferential location between the first set comprising 60 side wells. The first set and the second set of lateral wells are evenly distributed around the annular portion of the outer shell of the chip, the annular portion of the outer shell of the chip from at least 70% of the radius of the chip to the outer edge of the chip It can be extended.

The channels may extend radially from the center well to the side wells. The channels can only partially extend into the chip. The channels may have a width of about 30 μm. The channels may have a size and shape to accommodate movement of single cells from the central well to the lateral wells.

The MA chip includes relatively small μm-markers located inside at least a portion of the side wells to uniquely identify the side wells under microscope observation; And relatively large marks in mm located outside of at least a portion of the side wells so that the side wells can be uniquely identified by the naked eye.

The MA chip may further include a cap for injecting a cell suspension into the center well, and the cap may have a hole extending therethrough and may be disposed on the top of the center well.

The cap may be cylindrical, and may include a lip or shoulder that extends from the bottom surface of the cap into the center well, and the lip or shoulder can temporarily fix the cap during injection of cell suspension. The cap may be formed integrally with the chip.

According to some embodiments (examples) of the present invention, a method of isolating single cells from a cell suspension containing a small number of cells comprises a single central well and a plurality of lateral wells connected to the central well by a plurality of microchannels. Injecting a cell suspension containing a small number of cells into a containing microfluid fraction (MA) chip; And moving single cells to the lateral wells by a constant pressure driving flow through the microchannels.

The method can include identifying and isolating single or rare circulating tumor cells (CTCs) from the blood, and the CTC cells can be captured after a single or multi-step MA chip process. The method may include identifying and isolating single cells and objects from a complex matrix, the step of isolating a single object comprising bacteria, beads, particles, and viruses from the matrix, and separating Mycobacterium tuberculosis. And isolation of yeast cells of a particular genotype / phenotype. The method comprises identifying cells in the lateral wells; And recovering cells from the lateral wells containing only a single cell.

According to some embodiments (examples) of the present invention, a method for performing a single cell analysis includes a disk-shaped chip having a center and a surface; A single central well (entrance) located at the substantially center of the chip; A plurality of side wells (outlets) located in an annular portion of the outer shell of the chip; And providing a microfluid aliquot chip comprising a plurality of channels extending from the central well to the side wells. The method for performing the single cell analysis comprises injecting a cell suspension into an inlet of a microfluid fraction (MA) chip comprising a single central well and a plurality of lateral wells connected to the central well by a plurality of microchannels; And moving single cells to the lateral wells by a constant pressure driving flow through the microchannels. The method for performing the single cell analysis may further include adding a medium free of cells into the central well to generate positive hydrostatic pressure such that single cells remaining in the channels flow to the lateral wells.

The microfluidic fractions described herein can facilitate convenient, rapid, identifiable, and intact single cell isolation independent of cell size, morphology, and mobility. The isolated single cells can proliferate to form cloning within the lateral wells or can be easily recovered from the lateral wells as a single cell suspension of submicroliters for subsequent PCR and sequencing. Due to its high efficiency concentration ability, the method can also be applied to isolate single cancer cells from blood.

Dividing a confined liquid into multiple aliquots in the same volume using a single / multi-channel pipette for parallel analysis is a common experimental procedure in biological and chemical laboratories (eg cell suspension by manual / automatic pipettes) The serial dilution method, which is widely used to isolate single cells in cells. However, the difficulty in accurately identifying large amounts of aliquot volumes and single cells is limited to single cell genetic analysis and is very time consuming.

In contrast, microfluidic fractions described herein have significant advantages in reducing fraction volume and labor. Aliquots of multiple (e.g., 120) submicroliters can be well prepared within 1 minute, and single wells containing single cells can be quickly and accurately identified (e.g., at a rate of once per second) under normal inverted microscopy. Can be.

Microfluidic fractionation is a simple and effective strategy to quickly obtain aliquots of multiple submicroliters with minimal sample loss (about 18 nl per well, which may be substantially equal to a single microchannel volume). In addition, microfluidic fractionation is a simple, experience-independent method that does not require complex tools, additional training, or even background knowledge of microfluids.

In addition to the MA chips disclosed herein, equipment used in the process of the present invention, such as pipettes, petri dishes and optical microscopes, is readily available in conventional laboratories. During the entire process, including the injection of the original cell suspension and the movement of a single cell suspension, the liquid can be easily handled using a pipette, making it easy for beginners to use.

Some features and advantages of using the MA chip for single cell isolation are as follows:

-Easy operation. Both single cell suspension introduction and collection processes can be accomplished using only commonly used air displacement pipettes without requiring prior training.

-Fast sample injection. Dispensing a single cell suspension into 120 (one hundred twenty) 1 μl volume flank wells can take less than 1 minute.

-Easy single cell identification. Single cells in a 1.5 mm diameter lateral well can be identified very easily by a conventional optical microscope (1 well per second).

-Easy side well tracking. Each of the 1.5 mm diameter side wells is denoted by both a small microunit size for microscopic observation and a large macrounit size for visual observation.

-Compatibility with single cell PCR. Single cells can be separated into about 1 μL volume of liquid and transferred to vials using a pipette commonly used for subsequent single cell PCR and sequencing.

-High cell viability and cloning formation. The isolated single cells are healthy and can normally grow in lateral wells until cloning is formed.

-Selective single cell isolation. Single cells can be selectively isolated on the basis of size, suspension / adhesion morphology and fluorescence, such as when circulating tumor cells (CTCs) are isolated from the blood.

-High efficiency processing process. One MA chip provides 120 (one hundred twenty) flanking wells for single cell isolation and analysis.

High compatibility with cells of different sizes and shapes A single nucleated cell and a single prokaryotic cell having a diameter of 1 μm to 30 μm can be effectively separated into 1 μl volume lateral wells.

-Low cost. In mass production, the cost of each MA chip can be less than $ 1.

-Sterilization. The MA chip can be sterilized by standard methods such as sterilizer, alcohol immersion, and UV exposure.

Other features and aspects of the invention will be apparent from the following detailed description and accompanying drawings, which describe features according to embodiments of the invention, which are provided as an example. The above summary is not intended to limit the scope of the invention, and the scope of the invention is limited only by the appended claims.

One or more embodiments of the invention are described in detail with reference to the accompanying drawings. The accompanying drawings are provided for illustrative purposes only and represent typical or exemplary embodiments of the invention. These drawings are provided to aid the understanding of the present invention and should not be considered as limiting the scope or application of the present invention. It should be understood that, for clarity and ease of explanation, the drawings are not necessarily drawn to scale.
Some of the figures included in this specification show various embodiments of the invention observed at different viewing angles. In the following detailed description, drawings such as a plan view, a bottom view, or a side view are mentioned, but these drawings are for illustrative purposes only, and unless otherwise specified, imply or require that the present invention is implemented or used in a specific spatial direction. It is not done.
1 shows a top view of a microfluid fraction (MA) chip as an exemplary embodiment.
FIG. 1A shows a cross-sectional view of a portion of the MA chip of FIG. 1 taken along lines 1A-1A (the FIG. 1A view shows from the right edge of the MA chip to the slightly left portion of the center well).
1B is an enlarged plan view of a portion of the MA chip of FIG. 1.
1C is an enlarged plan view of a portion of the MA chip of FIG. 1.
2 shows a flow chart of a microfluid fractionation method.
The drawings are not intended to accurately implement or limit the invention in the form described. It should be understood that the invention may be practiced in modified or modified form and limited only by the claims and their equivalents.

In some cases, the present invention is described herein in connection with exemplary environments. Descriptions in these exemplary environments are provided to represent various features and embodiments of the invention in the context of exemplary applications. Those skilled in the art reading this description will clearly understand how the present invention can be implemented in different alternative environments.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

All patents, applications, and publications mentioned in this specification are incorporated by reference in their entirety. If the definitions set forth in this section are inconsistent or inconsistent with the definitions set forth in applications, publications, and other publications incorporated herein by reference, the definitions provided herein are in the documents incorporated herein by reference. Takes precedence over the definition of

Any dimension described herein is exemplary and should be regarded as an approximation, unless otherwise stated, and can be interpreted as indicating a relative measure of various elements that can be described.

In accordance with the present invention, a relatively large amount of cell suspension comprising multiple cells (e.g., 120 uL) is substantially simultaneous with multiple aliquots (e.g., 120) with single cells, typically in less than 1 minute. Can be uniformly distributed. Random and reliable single cell separation can be implemented using a microfluid aliquot chip (MA chip) comprising a single central well in fluid communication by several individual lateral wells and multiple individual microchannels. The MA chip can be used to effectively separate single cells in lateral wells from a small number of cells in the central well (eg, about 2 to 120 cells) by a constant pressure drive flow. The MA chip has high cell regeneration efficiency. Due to the rapid separation and easy identification of single cells, high cell viability, and convenient migration of single cell suspensions of submicroliters, the MA chip can be easily applied to single cell cloning, PCR, and sequencing.

Example of MA chip Concrete

1 shows a microfluidic fractionation (MA) chip 100 according to an embodiment of the present invention, and the microfluidic fractionation (MA) chip 100 may also be referred to as a “MA chip”. 1A shows a portion of the MA chip of FIG. 1. 1B shows a part (enlarged) of the MA chip of FIG. 1. FIG. 1C shows a part (enlarged) of the MA chip of FIG. 1.

The MA chip may include a thin sheet 102 made of a flexible or semi-rigid material such as polydimethylsiloxane (PDMS) having a thickness of about 1 mm. The MA chip may be in the form of a disc as a whole with a geometric center CL and a diameter of about 6 cm (radius of about 3 cm). The MA chip may have a total weight of about 1.7 g. The MA chip may have a size and shape that can be mounted on a Petri dish having a diameter of about 8.5 cm. The sheet forming the MA chip may include an upper surface 102a and a lower surface 102b respectively corresponding to upper and lower surfaces of the entire MA chip.

The MA chip may have a disk shape such as a polygon. Hereinafter, a disk-shaped MA chip is mainly described.

Hereinafter, various configurations (center well, side well, channel) extending on the upper and lower surfaces formed on the entire MA chip will be mainly described.

The MA chip is a plurality of channels 112a and 112b (collectively extending radially) from the central well 110 to the same plurality of lateral wells 114a and 114b (collectively referred to as "114"). (Also referred to as “112”) single (one) central well 110. The channels 112 are in fluid communication with the center well 110 and the side wells 114.

The center well 110 may be used as an inlet, and may be in the form of a round hole extending at least partially within the upper surface of the MA chip, for example, about 500 μm. 1A, the center well can extend to pass completely through the MA chip. The center well has a diameter of about 3 mm. The volume of the central well may be 4 μl. The center well may be disposed at the geometric center of the MA chip. The central well allows the user to access the upper surface of the MA chip and is used to inject a cell suspension into the MA chip.

The side wells (or outer wells 114) may be used as an exit, and may be in the form of a round hole extending at least partially into the upper surface of the MA chip, for example, about 500 μm. 1A, the side wells can extend to pass completely through the MA chip. The side wells may have a diameter of about 1.5 mm. The volume of the side wells may be 1 μl. The side wells may be distributed around the outer peripheral portion (or annular portion) of the MA chip. The lateral wells allow the user to access the upper surface of the MA chip and are used to recover cells isolated from the MA chip.

For example, 120 (one hundred twenty) side wells 114 may be included. The plurality of side wells are the first set 114a of the side wells disposed at the first radius r1 from the center of the MA chip and the second set of the second set of radiuses R2 from the center of the MA chip ( 114b). For example, the first set of lateral wells is arranged (distributed, distributed) 60 (sixty) evenly (every 6 degrees) around the circumference at a radius r1 of 25 mm from the geometric center of the MA chip. Can contain two side wells. And, the second set of side wells is 60 (sixty) sides arranged (distributed, distributed) evenly (every 6 degrees) around the circumference at a radius r2 of 28 mm from the geometric center of the MA chip. Wells. The first and second set of side wells may be interleaved so that the second set comprising the 60 side wells is disposed in a circumferential position between the first set comprising the 60 side wells. In this case, 120 side wells will be evenly arranged at an angle of 3 degrees to the outer circumferential portion of the MA chip (also referred to simply as "circumference"). The number of side wells is not limited to this, and more or less numbers of wells may be arranged differently from the above. In FIG. 1, only a portion of the side wells 114a and 114b are indicated by reference numerals for clarity. 1A and 1B generally refer to a side well 114 that may be one of the first side well sets 114a or one of the second side well sets 114b. 1C shows only four of the 120 side wells 114a and 114b, some of which are indicated by reference numerals.

The MA chip 100 may be in the form of a disk having a center and a radius of 3 cm. The center well may be disposed at the center of the disk. The lateral wells may be disposed in the “outer circumferential portion” (or “outer ring annular portion”) of the disc with radius portions 25 mm or 28 mm from the center of the disc. The annular portion of the outer shell of the disk (where the side wells are located) is at least about 50% (e.g., at least 60% or at least 70% or at least 75% or at least 80%) of the total radius r of the disk, or at least 90%, almost 100% at maximum), as part of the disk extending from the outer edge of the disk (100% radius (“r”)). See member 104 in FIG. 1C.

The outer peripheral portion (or region) of the outer shell of the MA chip may be annular. The annular is a ring-shaped region surrounded by two concentric circles. 1C shows a portion of the MA chip, the portion of which is in the form of a disk with a radius r (the center of the MA chip is not shown). The first side well set 114a is of the MA chip It can be arranged at a radius r1 from the center. The second side well set 114b may be disposed at a radius r2 from the center of the MA chip, where r2 is greater than r1 (r2> r1). The outer peripheral portion of the outer shell of the MA chip may extend from a radius r3 from the center of the MA chip to the outer edge of the MA chip located at a radius r from the center of the MA chip, and the radius r3 is It may be smaller than the radius r1 (r3 <r1).

The channels 112 (also referred to herein as “microchannels”) can be elongated with a length and width, for example at least 30 μm (only a fraction if the thickness of the chip is 1 mm). It may partially extend to the lower surface of the MA chip. This is shown in Figure 1a. The microchannels preferably extend at least partially on the MA chip, but may extend over the entire MA chip. The microchannels may have a width of 30 μm. The dimensions of the microchannels can be from 20 μm to 100 μm, and these dimensions are applicable in both depth and width.

The microchannels 112 may extend radially from the center well to the side wells, for example from the outer edge of the center well to the inner edge of the side wells. There can be one microchannel for each side well. Thus, for example, there may be 120 (one hundred twenty) microchannels extending from the single (one) central well to the 120 (one hundred twenty) lateral wells. The microchannels are generally provided for fluid communication and cell movement (movement) between the central well (entrance) and the lateral wells (outlets). The microchannels can be considered as channels, pipes, conduits, or connectors for connecting the outlets to the inlet. One end of each microchannel can be connected (in fluid communication) to the central well, while the other end of each microchannel can be connected (in fluid communication) to a given one of the plurality of lateral wells. have. Alternatively, the microchannels may extend in a shape other than a radial shape extending from the outer well to the side wells (eg, in the form of a spoke extending from the hub to the spokes).

Since the side wells can be arranged at different radii (r1, r2) from the center of the MA chip, the microchannels include 60 (sixty) microchannels extending into the first side well set 114a Can be arranged in a second set comprising 60 (sixty) microchannels extending into the first set 112a and the second side well set 114. The first microchannel set may have a length of about 23 mm. The second microchannel set may have a length of about 25 mm. Since the side wells are staggered (r1, r2, r1, r2), when the length between the side wells is long, the second microchannel set 112b is adjacent to two of the first side well sets 114. It may be necessary to pass between wells (if the length between the side wells is short, the first microchannel set 112a extends to the first side well set 114a, and it is not necessary to pass between the wells) ). For clarity, only a few microchannels 112a and 112b are shown in FIG. 1 with reference numerals. 1A and 1B generally mean a microchannel 112 that can be one microchannel of the first microchannel set 112a or one microchannel of the second microchannel set 112b. 1C shows only four of the 120 microchannels 112a and 112b, some of which are indicated with reference numerals.

The microchannels may have a width (or side length) of about 30 μm. The microchannels may all have the same width. The cross-sectional areas of the microchannels may be square, the width and depth may be similar, and may have straight sidewalls. The microchannels may have a tapered or semi-circular cross section. In general, the microchannels can be of a size and shape that can (and optionally facilitate) the migration (in the fluid) of single cells from the central well to the lateral wells. Each of the microchannels may have a volume of about 18 nl.

1A shows that the microchannels 112 are flat (eg, the MA chip is in a petri dish located on a flat table top). The microchannels can be inclined toward the side wells 114 to help positive pressure-driving fluid flow from the center well to the side wells. That is, the microchannel may have a shallower center well end and a deeper end at its side well. The width of the microchannel may be uniform or variable depending on the overall size (length) of the microchannel from the center well to the side well. In general, all of the microchannels on a given MA chip may have the same width and length as each other, or some microchannels may have different widths and depths (in FIG. 1, some of the microchannels 112a are different microchannels). Shown shorter than the channels 112b). MA chips can be fabricated to include microchannels with uniform widths (and depths) from one another. Although the MA chip in the figure is shown to include uniform microchannels, the MA chip can be fabricated to include microchannels of different sizes (ie, width and / or depth).

The cap 120 may be disposed on the center well 110. The cap may be in the form of a cylinder having an outer diameter of about 5 mm and an inner diameter (or through hole) of about 3.0 mm, and the cylinder may have a uniform bore from one end to the other. The length of the cap may be about 5 mm. The cap may be made of plastic such as PDMS separated from the MA chip 100, and may be a member used with the MA chip 100. The cap is shown in the figure as being spaced apart from the MA chip. The cap may have a volume of about 112 μl. The hole (bore) extending through the cap may have a diameter of about 1.5 mm. The cap may be provided integrally with the MA chip rather than as a separate member from the MA chip.

As shown in FIG. 1A, the cap 120 is placed on top of the central well 110 and contains a large number of small cells (eg, 1 μl) that are suspended in a well used for cloning or recovery. A pipette tip (not shown) containing a 120 μL cell suspension can be connected to a single central well to allow easy injection of large volumes (eg, 120 μL) of cell suspension into the lateral (eg, 120) side wells.

The upper and lower surfaces of the cap 120 are opened. The lower surface of the cap extends slightly in the upper portion of the center well to extend a lip or shoulder extending from the lower surface of the cap (as shown) to temporarily store the cell suspension injected during injection of the cell suspension. ). The cap is suitably placed over the central well and acts in the same way as a funnel (although not cylindrical, but cylindrical) to facilitate the injection of cell suspension into the central well. The top surface of the cap is not closed, and the cap is similar to chimney and functions as a guide and will be referred to as a "barrel", or "flue" or "duct" You can.

In order to identify the plurality of individual side wells and to easily distinguish them, an indicator may be provided on the top surface 102a of the MA chip. For example, relatively small micrometer (um) -unit numbers ranging from "1" to "120" correspond to the lateral wells under microscopic observation (with a unique identification number) and the MA chip adjacent to each of the individual lateral wells. It can be located on the upper surface (eg, radially / inside). The μ-unit markers are not shown in FIG. 1. Some of these μ-unit markers can be observed in FIG. 1C. Also, a relatively large set of millimeter (mm) unit numbers (or subsets), such as an even number in the range "2" to "120", is adjacent to the lateral wells (eg, out of radial / outside) In other words, from the center of the MA chip, radially outside of the second lateral well set 112b located at a larger radius r2) can be observed with the naked eye on the upper surface of the MA chip. So as to correspond to the side wells. Mm-unit marks of odd side wells 112a may be omitted from the product due to lack of space (or odd numbers in mm in the range of "1" to "119" are outside the first side well set 112a). Can be placed, and mm unit markers for even side wells can be omitted (or, if space is sufficient, a number in mm for all of the side wells can be included). These mm unit markers are shown in FIGS. 1A and 1C. The numbers in mm should correspond to the µm-unit numbers of the side wells provided above.Other indications that can identify and distinguish each of the side wells (eg, markers, letters, colors, symbols) All or some of these marks may be located on the underside of the MA chip to identify side wells, the side wells do not extend completely through the MA chip. It may be on the lower side of the cover for the well identified individual side-well locations.

Design and manufacture of the MA chip

The MA chip can be manufactured using conventional photolithography and polydimethylsiloxane (PDMS) molding techniques. PDMS belongs to a high molecular organosilicon compound commonly referred to as silicone. PDMS is the most widely used silicon-based organic polymer, and is known to have particularly unique rheological properties (or fluidity). PDMS is optically transparent, generally inert, non-toxic, and non-flammable. PDMS, also called dimethicone, is one of several silicone oils (polymerized siloxanes). PDMS is used in a variety of applications, from contact lenses and medical devices to elastomers.

The MA chip is designed using AutoCAD software (Autodesk), and can then be printed with a 4-inch glass photomask (Photo Sciences, Inc.). The PDMS MA chip can be manufactured by standard photolithography and elastomeric molding. In summary, SU-8 3025 negative photoresist (MicroChem Corp.) was spin-coated onto a 4-inch silicon wafer (Silicon Quest International Inc.) at 2000 rpm (Laurell Technologies Corp.) for 1 minute. ) To form a 30 μm thick sheet. After baking, UV exposure, and development, mixed polydimethylsiloxane (PDMS; 10A: 1B; Dow Corning Corp.) was poured onto the photoresist and heated at 80 ° C for 25 minutes can do. After curing, the 1 mm thick PDMS sheet can be peeled off and punched using a punch (Ted Pella, Inc). The round PDMS MA chip can be automatically sealed into a commercially available Petri dish (Fisher Scientific) with a diameter of 8.5 cm on the market without generating air bubbles. If necessary, the MA chip can be sterilized using a standard autoclave.

In addition to PDMS, MA chips can be made of other materials including poly (methyl methacrylate), polystyrene (PS), and polycarbonate (PC).

PMMA, also known as acrylic or acrylic glass, is also known under the trade names Plexiglas, Acrylite, Lucite, and Perspex, and is a transparent thermoplastic material often used in the form of a light sheet or as a glass substitute due to its anti-scattering function.

PS is an aromatic synthetic polymer made of styrene monomer. PS can be solid or foamed. Universal PS is transparent, hard, and fragile. The unit weight price of PS is cheap. PS has a rather poor defense against oxygen and water vapor and has a relatively low melting point. PS is one of the most widely used plastics.

PC is a group of thermoplastic polymers containing carbonate groups in its chemical structure. Polycarbonates used in engineering are strong and rough materials, some of which are optically transparent. The PC can be easily processed, molded, and thermoformed. Due to these properties, polycarbonate is applied in many fields.

Isolation and recovery of single cells

2A-2E show one embodiment of a microfluid fractionation method 200 performed to isolate and recover single cells using the MA chip 100 described above. The method or process consists of sequentially steps. Some of these steps, or some of the steps, may be performed in a different order than what is described herein. The estimated time to perform each of the above steps can be mentioned.

Step 1 (202, assembly):

The MA chip 100 is disposed in a Petri dish (not shown, Appendices). The MA chip 100 may have a diameter of 6 cm, and the Petri dish may have a diameter of 8.5. Due to the excellent elasticity and flexibility of the thin PDMS sheet, the MA chip can quickly recover a flat shape even after being rolled, and then can be sealed in close contact with a hard, flat surface such as the bottom surface of a petri dish without air bubbles. This step is expected to take 0.1 minutes (6 seconds) to complete.

Step 2 (204, capping):

The cap 120 may be disposed on the upper surface 100 of the MA chip and on the center well 110 (see FIG. 1A). The cap may be in the form of a cylinder. The diameter of the center well may be 3 mm. The cap may have an OD of 5 mm (sufficient to overlap / greater than the center well), and the hole extending through the cap may have a diameter of 1.5 mm. The holes in the cap can be well-aligned over the center well, making it possible to inject the cell suspension into the center well using a pipette. The cap may have a height of 5 mm. The PDMS cap placed over the center well facilitates the injection of liquid (cell suspension) into the MA chip using a pipette. This step is expected to take 0.1 minutes (6 seconds) to complete.

The PDMS cap includes a through hole having a diameter of 1.5 mm and a depth of 5 mm. This size is well compatible with 100 to 1000 μl pipette tips, allowing injection of liquid into the MA chip without leakage.

Step 3 (206, preparative):

To collect a 120 μl cell suspension (not shown), inject into the central well (entrance) of the MA chip through the cap using a pipette with a 100 to 1000 μl tip (not shown, see Appendix). This phase is expected to take 1 minute (60 seconds) to complete.

Due to the uniformly distributed pressure around the inlet, the cell suspension can be distributed substantially simultaneously and uniformly into the large number (e.g., 120) side wells 114, resulting in random and reliable single cell separation. Is made. A constant pressure drive flow pushes single cells from the central well (entrance) through the microchannels 112, 112a, 112b to the lateral wells (exits, 114 (114a, 114b)) (transport, transfer, transfer) This can be referred to as "microfluid fractionation."

After the aliquot, 100 μl of cell-free medium is added dropwise into the central well to push out potential single cells in the microchannels (1.8% probability) to the outlets (lateral wells). That is, after injecting 120 μl of cell suspension into the MA chip using a pipette, one single cell may occasionally remain in the microchannels (1.8% probability). By injecting 100 μl of cell-free medium into the center well, positive hydrostatic pressure will be created from the center well to the lateral wells. This positive hydrostatic pressure will push the remaining single cells into the lateral wells.

Step 4 (208, identification):

Single cells in the lateral wells 114 may be identified by an inverted microscope at a rate of 1 well / s, and then recorded in micrometer-scale numbers located inside the lateral wells and specifically designed for microscopic observation. Because the area of an inverted microscope equipped with an X10 objective lens (having a diameter of 2 mm) is larger than the size of a MA chip well (having a diameter of 1.5 mm), only single lateral wells containing only single cells can be quickly differentiated. You can. It is expected that it will take 1 second for each well or a total of 2 minutes (120 seconds) for the entire well (120 side wells) to complete this step.

The distribution of 123 isolated single cells into wells indicates that almost all single cells were located in the direction of microchannel extension under the influence of microfluids. That is, under the influence of positive pressure from the central well to the lateral wells, all single cells can finally flow into the lateral wells. The exact location of each single cell in each 1.5 mm diameter lateral well may differ from well to well. However, it is also possible that the cells are located in the intersecting region of the microchannel and the lateral wells (the intersection, i.e., the radially innermost region of the lateral well just beyond the end of the microchannel connected to the lateral well).

Step 5 (210, recovery):

The desired single cells are inserted into the lateral wells from 0.1 to 2.5 µl, according to even numbers in millimeters located outside of the lateral wells 114b and suitable for visual observation. It can be conveniently and directly recovered using a pipette (not shown).

Finally, cells were collected in a Petri dish, prepared in cell suspension, counted using a microscope, injected into a MA chip, and recovered as a single cell suspension using an air displacement pipette (Eppendorf). .

The entire process of obtaining single cells using the MA chip of the present invention has been described above. The process of preparing a single cell suspension having a known number of cells injected into the MA chip includes the following four steps. 1) Using a pipette, 1 μl of a single cell suspension is added dropwise to a Petri dish. 2) Count cells using an inverted microscope. 3) 119 μl of cell-free medium is added to 1 μl of the single cell suspension. 4) The entire single cell suspension with a known cell number is recovered to collect a small amount of fluid. Thereafter, steps 1 to 5 (assembly, capping, aliquot, identification, and recovery) described above may be performed.

Instead of a cap, a PDMS (or other material) mat may be placed on the MA chip (which may be referred to as "MA dish"). The thickness of the mat is 5 mm, and may have a 1.5 mm diameter hole in the center of the mat. As an example procedure, the following steps can be performed.

1) A 120-well MA dish is placed on the surface of a tissue culture dish.

2) Place the mat on the MA dish. The 1.5 mm diameter hole is arranged to align vertically with the 3 mm central well.

3) Using a pipette tip, a single cell suspension is aspirated at a volume exceeding 120 μl to a concentration of less than 1 cell / ul.

4) Insert the pipette tip into the mat hole.

5) Inject a single cell suspension into the 120 side wells by pushing the button of the pipette downward.

6) Each lateral well can be filled with 1 μl of cell suspension within 30 seconds.

7) Remove the pipette tip and mat.

8) Identify each lateral well, mark all lateral wells containing single cells, and transfer single cells to a vial using a pipette for subsequent PCR experiments.

9) Alternatively, the single cells can be cultured as they are in side wells to achieve cell cloning.

Comments and conclusions

Microfluid fractionation techniques, functionality and applicability for the isolation of various single cells, regardless of cell size, morphology and mobility, were described. Microfluidic fractionation involves single cells from millions of cells with the potential to be used to separate circulating tumor cells (CTCs) from a small number of cells and blood from critically important clinical samples with limited cell counts. It is possible to selectively separate. In addition, the microfluid fraction is very well compatible with single cell replication, where the viability of a single cell is well maintained and single cell PCR capable of preparing a 40 nanoliter single cell suspension within 10 minutes. In addition, the MA chip is low cost, equivalent to the cost of 1.7 g PDMS (about $ 0.20), requires no junctions and valves, and is easy to mass-produce, making it readily available to single-cell researchers in a typical laboratory.

Features such as high-speed aliquots and 120 aliquots per operation allow a single MA chip to have 120 enrichment factors (EFs). Therefore, in theory, one target cell can be successfully separated from millions of cells through 3 to 4 MA chips, and the corresponding EFs may be 1.7Х10 ⁶ and 2.1Х10 ⁸ , respectively. Therefore, the MA chip has high cell regeneration efficiency.

The MA chip can effectively separate (isolate) single cells from a small number of cells. Different MA chips with different sized microchannels consisted of a single germination yeast with a diameter of 4 μm, a single floating T cell with a diameter of 8 μm, a single MDA-MB-231 breast cancer cell with a diameter of 16 μm, and a length of 22 μm and a width of 3 μm. The mobility of Trypanosoma brucei (Trypanosoma brucei), such as the size, shape, and mobility of the cell, regardless of the type of cell is applicable.

The size of the channel, including width and depth, must be greater than the diameter of the cell. This can reduce cell damage during migration within the micro-channel-cells can easily migrate from the central well to the lateral wells. In this case, the width and depth of the microchannels are both 30 μm, which is larger than the diameter of the cells tested. In general, microchannels with dimensions (width and depth) greater than the cell diameter can be used.

Even if there are a small number of cells (eg 2 to 20 cells) in the original suspension, the MA chip can separate single cells.

If the damage in cell viability is negligible and the cells in suspension do not show a morphologically apparent difference, the MA chip can be used to selectively separate single cells based on their adherence. Although there is negligible lysate loss due to the static pressure driven flow, the desired cells can be recovered by adding a small amount of trypsin to the wells where they are present or directly lysed under adherence to study the genotype-phenotype correlation at the single cell level. It offers potential possibilities.

Due to features such as rapid separation and easy identification of single cells, high cell viability, and convenient migration of single cell suspensions of submicroliters, the MA chip is well compatible with single cell cloning, PCR and sequencing. Wide type (WT) and PTEN knock out (KO) SUM 159 cells were used as proof of concept experiments. PTEN KO cells were generated by transient transfection with plasmids encoding sgRNA targeting PTEN (phosphatase and tensin homologs) and Cas9 protein into SUM 159 cells. After single cell separation by MA chip, the cells proliferated normally, and the number of cells at a given time point was measured. As a result, it was shown that PTEN KO cells had an average of 2 times shorter than 28.3 hours of WT cells at 25.9 hours. These results are consistent with previous studies that PTEN deficiency can promote cell proliferation.

The methods and apparatus described herein can be used to identify and separate single cells (eg, single CTC cells from blood) from complex matrices. Specifically, CTC cells can be captured in lateral wells after a single or multi-step MA chip process. The methods and devices disclosed herein can be used to separate single cells and objects from complex matrices (e.g., separate single objects, including bacteria, beads, particles, viruses, from the matrix) , Mycobacterium tuberculosis), can be used to isolate single clones (eg yeast cells of a particular genotype / phenotype).

Difference from US Patent No. 6,632,656

The MA chip 100 of the present invention has some similarities to the rotatable disk 18 of US Pat. No. 6,632,656 ("Thomas") described above. Both inventions are of the general disc shape. The channels of US Pat. No. 6,632,656 (“Thomas”) have a width of 20 to 30 μm. The microchannels 112 of the present invention may have a width of about 30 μm.

Specifically, the MA chip 100 of the present invention can be distinguished by the following points from the rotatable disk 18 of US Pat. No. 6,632,656 ("Thomas"):

-In U.S. Patent No. 6,632,656 ("Thomas"), the chambers 2, 3, 7 are shown as being located in the central portion of the disc. A waste channel (10) occupies the outer portion of the disk. The side wells 114, 114a, 114b of the MA chip described herein are located in the outer portion of the MA chip.

-In the above U.S. Patent No. 6,632,656 ("Thomas"), the channel 6 is an inlet channel 1, a subsequently connected chamber 2, another connected channel 4, a subsequently connected chamber 3, and subsequently connected It includes an outlet channel 5 and a waste channel 10 connected subsequently. The channels 112, 112a, 112b of the MA chip described herein extend from the center well (entrance) to the side wells (outlets), and any member (another chamber) between the center well and the side well. Or wells).

-In the U.S. Patent No. 6,632,656 ("Thomas"), cells in a channel are moved from chamber to chamber using rotation and centrifugal force. In the process 200 described herein, cells are moved from the central well (entrance) to the lateral wells (outlets) using the MA chip described herein, mainly depending on the positive pressure.

-The chambers of U.S. Patent No. 6,632,656 ("Thomas"), characterized in that the cells move by centrifugal force, are closed. The lateral wells described herein, characterized in that the cells mainly migrate by static pressure, are open (accessible through the top surface of the MA chip).

-In the device of U.S. Patent No. 6,632,656 ("Thomas"), liquid and cells flow out of the device through channels. However, for the MA chip described herein, all liquid containing cells injected through the inlet (center well) can be collected completely and stored in the outlets (side wells). That is, in the case of the present invention, the liquid does not flow out of the device, and the method of collecting the sample is completely different from the device of US Pat. No. 6,632,656 ("Thomas").

-The MA chip described herein is focused on an aliquot (the sample is divided into many aliquots having the same volume, and there is no loss of the sample). In contrast, the device of US Pat. No. 6,632,656 ("Thomas") focused on filtration, trapping of cells into closed chambers, and liquid spillage out of the device.

-U.S. Patent No. 6,632,656 ("Thomas") injects liquid into the device using the centrifugal force provided by a specific centrifuge. On the other hand, the present invention easily injects a liquid such as a cell suspension into the MA chip using only a general air displacement pipette (used in all laboratories). This will greatly reduce the dependence on external devices such as centrifuges.

-The diameter of the side wells described herein is about 1.5 mm. This size is important for microscopic identification of single cells in the lateral wells and recovery of liquid in the lateral wells using a 0.1 to 2.5 μl pipette tip. Since the area of an inverted microscope equipped with an X10 objective lens (2 mm in diameter) is larger than the size of a MA chip well (1.5 mm in diameter), only single wells containing only a single cell can be quickly distinguished (1 well / s). ). The chambers 2, 3, 7 of the above US Pat. No. 6,632,656 (“Thomas”) are significantly larger than the side wells of the MA chip of the present invention. In U.S. Patent No. 6,632,656 ("Thomas"), "circular 'wells' having a diameter of about 400 to 600 μm are described. In addition, in U.S. Patent No. 6,632,656 (" Thomas "), 50 μm or 100 μm A channel with a depth of 100 μm and a width of 100 μm is described, which is considerably larger than the microchannels described herein (in particular, the cross-sectional area associated with the flow of fluid is quite large).

-The device of U.S. Patent No. 6,632,656 ("Thomas") is mainly used to grow and analyze cells (eg, drug screening). The device is subject to a large number of cells, and the goal is drug screening based on a large number of cells. The MA chip described herein is mainly used to select or isolate single cells, the target sample is individual cells, and the purpose is to perform single cell based analysis including single cell cloning, PCR, and sequencing.

-The MA chip described herein has excellent ability to separate or select target cells from a cell population, such as the selection and isolation of single CTC cells (circulatory tumor cells) from blood. In contrast, the device of U.S. Patent No. 6,632,656 ("Thomas") does not have this function and cannot separate target cells (no function to separate individual cells).

Although the invention has been described above by means of various exemplary embodiments and embodiments, the various features, aspects, and functions described in each of the one or more embodiments are not limited to the specific embodiments in which they are described. , Regardless of whether the embodiments are described and whether features have been described as part of the described embodiments, may be applied alone or in combination to one or more other embodiments. Accordingly, the scope and scope of the invention should not be limited by any of the exemplary embodiments described above.

Claims (20)

A disk-shaped chip having a radius, center, upper surface, lower surface, outer edge, and thickness;
A single central well (entrance) disposed substantially at the center of the chip, extending to the top surface of the chip, and accessible from the top surface of the chip;
A plurality of side wells (outlets) disposed in an annular portion of the outer shell of the chip, extending to the upper surface of the chip, and accessible from the upper surface of the chip; And
And a plurality of channels extending from the bottom surface of the chip and extending from the center well to the side wells in fluid communication with the center well and the side wells. Microfluid fraction (MA) chip. According to claim 1,
The chip is thin made of a flexible or semi-rigid material selected from the group consisting of polydimethylsiloxane (PDMS), poly (methyl methacrylate) (PMMA), polystyrene (PS), and polycarbonate (PC). A microfluid fractionation (MA) chip for single cell separation, comprising a sheet. According to claim 1,
The chip,
Thickness of 1 mm;
Size and shape that can be mounted in a Petri dish; And
Radius of 3 cm; Microfluid fractionation (MA) chip for single cell separation, characterized in that it has at least one of the characteristics. According to claim 1,
The center well extends completely through the chip; And
The side wells extend to pass completely through the chip; Microfluid fraction (MA) chip for single cell separation, characterized in that. According to claim 1,
The center well,
Diameter of 3 mm; And
Volume of 4 μl; Microfluid fractionation (MA) chip for single cell separation, characterized in that it has at least one of the characteristics. According to claim 1,
Each of at least some of the side wells,
Diameter of 1.5 mm; And
Volume of 1 μl; Microfluid fractionation (MA) chip for single cell separation, characterized in that it has at least one of the characteristics. According to claim 1,
The first set of plurality of side wells is located at a first radius r1 from the center of the chip; And the second set of the plurality of side wells is located at a second radius (r2) from the center of the chip, the second radius (r2) is a single cell separation, characterized in that greater than the first radius (r1) Microfluid fraction (MA) chip. The method of claim 7,
The first set and the second set of lateral wells are interleaved such that the second set of lateral wells is located at a circumferential location between the first set comprising 60 lateral wells. Separation microfluid fraction (MA) chip. The method of claim 7,
A microfluidic fractionation (MA) chip for single cell separation, characterized in that the first set and the second set of sidewalls are evenly distributed around the annular portion of the outer shell of the chip. According to claim 1,
A microfluidic fractionation (MA) chip for single cell separation, characterized in that the annular portion of the outer shell of the chip extends from at least 70% of the radius of the chip to the outer edge of the chip. According to claim 1,
The channels,
Radially extending from the center well to the side wells;
Only partially extending into the chip;
Width of 30 μm; And
Size and shape to accommodate movement of single cells from the central well to the lateral wells; Microfluid fractionation (MA) chip for single cell separation, characterized in that it has at least one of the characteristics. According to claim 1,
The chip,
Relatively small μm-markers located inside at least a portion of the lateral wells to uniquely identify the lateral wells under microscopic observation; And
Microfluidic fractionation for single cell separation (MA) further comprising; relatively large mm unit markers located outside of at least a portion of the side wells so that the side wells can be uniquely identified by the naked eye. chip. According to claim 1,
The chip further includes a cap for injecting a cell suspension into the central well, the cap has a hole extending therethrough, and a microfluid fraction for single cell separation (MA) characterized in that it is disposed on the top of the central well. ) Chip. The method of claim 13,
The cap is cylindrical, and includes a lip or shoulder that extends from the lower surface of the cap into the central well, wherein the lip or shoulder temporarily fixes the cap during injection of cell suspension. Microfluid fraction (MA) chip. The method of claim 13,
The cap is a microfluidic fractionation (MA) chip for single cell separation, characterized in that formed integrally with the chip. Injecting a cell suspension containing a small number of cells into a microfluid fraction (MA) chip comprising a single central well and a plurality of lateral wells connected to the central well by a plurality of microchannels; And
And moving single cells to the lateral wells by a constant pressure driving flow through the microchannels. delete The method of claim 16,
The above method,
Identifying cells in the lateral wells; And
A method of isolating single cells from a cell suspension containing a small number of cells, further comprising the step of recovering cells from the lateral wells containing only a single cell. A disk-shaped chip having a center and a surface;
A single central well (entrance) located at the substantially center of the chip;
A plurality of side wells (outlets) located in an annular portion of the outer shell of the chip; And
And providing a microfluid fraction chip comprising a plurality of channels extending from the center well to the side wells.
Injecting a cell suspension into the inlet of a microfluid fraction (MA) chip comprising a single central well and a plurality of lateral wells connected to the central well by a plurality of microchannels; And
The method of performing a single cell analysis, characterized in that further comprising; moving the single cells to the lateral wells by a constant pressure driving flow through the microchannels, The method of claim 19,
The method further comprises the step of adding a cell-free medium into the central well to generate positive hydrostatic pressure such that single cells remaining in the channels flow to the lateral wells. Way.

Images